Development of a real-time PCR-based method for detection of Xylophilus ampelinus |
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| Authors: | T Dreo K Gruden C Manceau J D Janse M Ravnikar |
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| Institution: | National Institute of Biology, Department of Plant Physiology and Biotechnology, Vežna pot 111, 1000 Ljubljana, Slovenia;;Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia;;UMR Pathologie Végétale, INRA-INH-Universitéd'Angers, Institut National de la Recherche Agronomique, Centre d'Angers, F-49071 Beaucouzé, France;and;Plant Protection Service, Department of Bacteriology, Geertjesweg 15, 6706 EA Wageningen, Netherlands |
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| Abstract: | A real-time PCR MGB-probe-based detection method specific to Xylophilus ampelinus , the cause of grapevine bacterial blight, was developed. Used in combination with the DNeasy plant mini kit, the sensitivity of X. ampelinus detection was approximately 100 cells from tissue extracts, surpassing the sensitivity of an existing nested PCR method at least tenfold. In field samples a high correlation was observed between real-time PCR cycle threshold (Ct) values obtained and X. ampelinus isolation on artificial media. Isolation was successful from samples with Ct values below 25. Lower concentrations of X. ampelinus , with Ct values up to 36, could also be reliably detected in real-time PCR. The newly developed method offers a reliable and sensitive test for X. ampelinus, suitable as a screening test, complementary to isolation on media or other methods, and could also be used for fast and specific identification of isolated colonies and for relative quantification of X. ampelinus bacteria. |
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| Keywords: | grapevine bacterial blight Vitis vinifera |
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